2-Keto-L-gulonic acid is a significant intermediate in the preparation of L-ascorbic acid (vitamin C), an essential nutrient. While 2-keto-L-gulonic acid has been synthesized in the past on an industrial scale using the Reichstein method (Helvetica Chimica Acta 17:311 (1934)), fermentation processes employing one or more microorganisms are preferred for commercial production of 2-keto-L-gulonic acid. U.S. Pat. No. 2,421,611, for example, discloses a method involving microbial oxidation of D-glucose to 5-keto-D-gluconic acid, followed by chemical or microbial reduction to L-idonic acid and subsequent microbial oxidation to 2-keto-L-gulonic acid. Japanese Patent Publication Nos. 39-14493, 53-25033, 56-15877 and 59-35290, for example, disclose similar processes involving the microbial oxidation of D-glucose to 2,5-diketo-D-gulonic acid followed by microbial or chemical reduction to 2-keto-L-gulonic acid. Fermentative pathways involving oxidation of L-sorbose to 2-keto-L-gulonic acid via a sorbosone intermediate have also been developed, using Gluconobacter oxydans (U.S. Pat. Nos. 4,935,359; 4,960,695; and 5,312,741), Pseudogluconobacter saccharoketogenes (U.S. Pat. No. 4,877,735), Pseudomonas sorbosoxidans (U.S. Pat. Nos. 4,933,289 and 4,892,823), and mixtures of microorganisms (U.S. Pat. Nos. 3,912,592; 3,907,639; and 3,234,105).
Prior to conversion into ascorbic acid, however, 2-keto-L-gulonic acid must first be isolated from the fermentation broth. As described in U.S. Pat. No. 4,990,441, for example, 2-keto-L-gulonic acid can be recovered from a fermentation broth by a process comprising the steps of: (a) removing insoluble material from the broth by centrifugation, filtration in the presence of a flocculating agent, or ultrafiltration; (b) removing inorganic cations by acidification; and (c) isolating 2-keto-L-gulonic acid by crystallization. High yields of 2-keto-L-gulonic acid are difficult to obtain by this method, however, due to the high solubility of 2-keto-L-gulonic acid in the crystallization mother liquor.
Moreover, conversion to ascorbic acid according to the method described in U.S. Pat. No. 5,391,771 includes preparation of the methyl ester of 2-keto-L-gulonic acid by reaction with methanol. This esterification reaction involves an equilibrium between the free acid form and methyl ester of 2-keto-L-gulonic acid and so requires substantially anhydrous conditions to ensure sufficient yields of the desired product for commercial application.
Accordingly, there remains a need for a process for recovering 2-keto-L-gulonic acid from aqueous solutions, such as fermentation broths, in high yields.
Processes for removal of acids from aqueous media have been developed employing anionic ion exchange resins. Such processes are described, for example, in U.S. Pat. Nos. 5,278,339 (cyclohexanedicarboxylic acids) and 4,323,702 (carboxylic acids such as adipic acid) and in International Publication No. WO 92/16490 (citric acid). In all of these processes, the acid of interest is first adsorbed onto an anionic resin to a maximum value (as determined by measuring the change in the concentration of acid in the effluent during loading) and then subsequently desorbed with an agent that regenerates the anionic resin. These processes are generally not, however, suitable for recovering 2-keto-L-gulonic acid from aqueous solutions because the water content of the recovered 2-keto-L-gulonic acid tends to be too high for subsequent esterification without additional purification steps.
Accordingly, there remains a need for a process for recovering 2-keto-L-gulonic acid from aqueous solutions, such as fermentation broths, substantially water-free.